Liver transplantation, with lifelong immunosuppression, remains the treatment of choice for patients with a variety of acute and chronic liver diseases. Immunosuppression, however, is associated with deleterious side effects including chronic rejection and an increased incidence of infection and malignancy. Thus, a major goal in clinical transplantation is to eliminate the need for immunosuppression and to induce donor-specific tolerance to the allograft. Tolerance induction strategies have show promise, however there are major gaps in our understanding of the underlying mechanisms that determine tolerance induction. Furthermore, the impact of viral infection on the alloimmune response and the induction and maintenance of tolerance are poorly understood. In preliminary studies, a rat orthotopic liver transplant model and total lymphoid irradiation (TLI), was used to induce tolerance induction to a liver allograft. In this model tolerance is associated with early apoptosis of graft infiltrating cells and increases in CD4+CD25+Foxp3+ T regulatory cells (Treg) in both the graft and periphery. Adoptive transfer of splenocytes from tolerant liver allograft recipients prolonged allograft survival and this effect was dependent on the presence of CD4+CD25+ T cells. The hypothesis to be tested is that early deletion of host T lymphocytes through apoptosis facilitates the emergence of CD4+CD25+Foxp3+ Treg cells that are critical to establishing donor-specific tolerance following TLI. We further hypothesize that concomitant viral infection can augment alloimmune responses in liver allograft recipients that can perturb immune regulatory mechanisms that promote tolerance. In Specific Aim 1 the mechanisms involved in the establishment and maintenance of tolerance in liver allograft recipients will be established. Experiments will be performed to determine if apoptosis is required for expansion of Treg and tolerance induction. Other factors that may participate in establishment of tolerance, including chimerism, are also studied. In Specific Aim 2 the goal is to determine the impact of viral infection on alloreactivity and tolerance induction in liver allograft recipients. Rat cytomegalovirus (RCMV) is used as a model virus to examine how active, latent, or chronic viral infection alters alloreactivity and graft survival. The influence of RCMV on deletion of host lymphocytes and development and function of regulatory mechanisms is determined. Finally, the effect on RCMV infection on established tolerance is assessed. These studies will provide a new understanding of immune tolerance and host-pathogen interactions in the context allogeneic transplantation.